Circulation and overwintering of Usutu virus lineages in north-eastern Spain: A one health perspective (2021-2025).

Usutu virus (USUV) is a mosquito-borne flavivirus that has expanded widely across Europe. While it has been traditionally considered an animal pathogen, reports of human infections and the detection of USUV in blood donors in Europe (including Spain) have increased the public health awareness of this emerging pathogen. In the last decade, serological evidence of USUV circulation in animals have been reported in Northeastern Spain (Catalonia) where human infections have also been recently detected; however, molecular confirmation of viral circulation in wild birds was still lacking. Our main goal was to investigate USUV circulation in Catalonia (2021-2025) using One Health approach through the West Nile virus surveillance program. Once USUV was detected, we aimed to assess USUV overwintering, genetic diversity and pathology in positive wild birds. From 369 bird samples collected, six blackbirds from the Barcelona province resulted USUV-positive in 2022, 2024, and 2025, showing signs of systemic infection. Additionally, USUV was found in a <i>Culex pipiens</i> mosquito pool, and sentinel and backyard birds showed serological evidence of flavivirus exposure in Girona province. Phylogenetic analyses revealed USUV overwintering, evidence of human spillover and the co-circulation of two distinct lineages, Africa 3 and Europe 2. Notably, Europe 2 was detected for the first time in Spain. These findings prove active enzootic USUV circulation in Northeastern Spain and highlight the need for One Health surveillance to support early USUV detection to reduce the risk of USUV transmission from enzootic cycle.